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Novel role of FATP1 in mitochondrial fatty acid oxidation in skeletal muscle cells.

Sebastián D, Guitart M, García-Martínez C, Mauvezin C, Orellana-Gavaldà JM, Serra D, Gómez-Foix AM, Hegardt FG, Asins G - J. Lipid Res. (2009)

Bottom Line: The cooverexpression of FATP1 and CPT1 also enhanced mitochondrial fatty acid oxidation, similar to the cooverexpression of FAT/CD36 and CPT1.However, etomoxir, an irreversible inhibitor of CPT1, blocked all these effects.These data reveal that FATP1, like FAT/CD36, is associated with mitochondria and has a role in mitochondrial oxidation of fatty acids.

View Article: PubMed Central - PubMed

Affiliation: Departments of Biochemistry and Molecular Biology, School of Pharmacy, University of Barcelona, Barcelona, Spain.

ABSTRACT
Carnitine palmitoyltransferase 1 (CPT1) catalyzes the first step in long-chain fatty acid import into mitochondria, and it is believed to be rate limiting for beta-oxidation of fatty acids. However, in muscle, other proteins may collaborate with CPT1. Fatty acid translocase/CD36 (FAT/CD36) may interact with CPT1 and contribute to fatty acid import into mitochondria in muscle. Here, we demonstrate that another membrane-bound fatty acid binding protein, fatty acid transport protein 1 (FATP1), collaborates with CPT1 for fatty acid import into mitochondria. Overexpression of FATP1 using adenovirus in L6E9 myotubes increased both fatty acid oxidation and palmitate esterification into triacylglycerides. Moreover, immunocytochemistry assays in transfected L6E9 myotubes showed that FATP1 was present in mitochondria and coimmunoprecipitated with CPT1 in L6E9 myotubes and rat skeletal muscle in vivo. The cooverexpression of FATP1 and CPT1 also enhanced mitochondrial fatty acid oxidation, similar to the cooverexpression of FAT/CD36 and CPT1. However, etomoxir, an irreversible inhibitor of CPT1, blocked all these effects. These data reveal that FATP1, like FAT/CD36, is associated with mitochondria and has a role in mitochondrial oxidation of fatty acids.

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Palmitate oxidation in mitochondrial fractions. Mitochondrial fractions that were obtained from cells infected with Ad-LacZ, Ad-FATP1, Ad-CPT1A, or Ad-FAT/CD36 were incubated for 1 h with agitation in 400 μl of pregassed complete MKRH buffer and 50 μl of a 2.5 mM 5:1 palmitate-BSA complex containing 10 μCi/ml [1-14C]palmitic acid, and palmitate oxidation to CO2 (A) and to ASPs (B) was measured. The ratio of ASPs/CO2 was also calculated (C). Data are the mean SE of five experiments performed in duplicate. *P < 0.01 versus Ad-LacZ, **P < 0.001 versus Ad-LacZ, &P < 0.001 versus the overexpression of only one protein (CPT1A, FATP1, or FAT/CD36), and #P < 0.01 versus control without etomoxir.
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fig3: Palmitate oxidation in mitochondrial fractions. Mitochondrial fractions that were obtained from cells infected with Ad-LacZ, Ad-FATP1, Ad-CPT1A, or Ad-FAT/CD36 were incubated for 1 h with agitation in 400 μl of pregassed complete MKRH buffer and 50 μl of a 2.5 mM 5:1 palmitate-BSA complex containing 10 μCi/ml [1-14C]palmitic acid, and palmitate oxidation to CO2 (A) and to ASPs (B) was measured. The ratio of ASPs/CO2 was also calculated (C). Data are the mean SE of five experiments performed in duplicate. *P < 0.01 versus Ad-LacZ, **P < 0.001 versus Ad-LacZ, &P < 0.001 versus the overexpression of only one protein (CPT1A, FATP1, or FAT/CD36), and #P < 0.01 versus control without etomoxir.

Mentions: To assess the role of FATP1 in fatty acid oxidation, we measured palmitate oxidation in mitochondria-enriched fractions. Palmitate oxidation to CO2 increased 1.7-fold in mitochondria isolated from FATP1-overexpressing cells compared with Ad-LacZ infected cells (0.16 ± 0.009 nmol palmitate/mg protein × h; Fig. 3A). As a positive control, we also evaluated palmitate oxidation in CPT1-overexpressing cells. Overexpression of CPT1A (3-fold increase in protein levels; Fig. 1C), the endogenous CPT1 isoform expressed in these cells (22), produced a 2.6-fold increase in mitochondrial palmitate oxidation to CO2. Moreover, overexpression of FAT/CD36 also increased palmitate oxidation to CO2 by 2.0-fold. To further evaluate the role of FATP1 in fatty acid oxidation, we overexpressed both FATP1 and CPT1 proteins together. The overexpression of CPT1A and FATP1 enhanced fatty acid oxidation to CO2 by 4.7-fold, showing an additive effect (Fig. 3A). Similarly, cooverexpression of CPT1A and FAT/CD36 also produced an additive effect on palmitate oxidation to CO2 (4.1-fold increase). Palmitate oxidation to ASPs, essentially consisting of acyl-carnitines, Krebs cycle intermediates, and acetyl-CoA, was increased from 1.4- to 1.8-fold in CPT1A-, FAT/CD36-, FATP1-, CPT1A+FAT/CD36-, and CPT1A+FATP1-overexpessing cells compared with Ad-LacZ control cells (36.1 ± 1.5 nmol palmitate/mg protein × h; Fig. 3B). When mitochondria were incubated with etomoxir, an irreversible inhibitor of CPT1, the increments in fatty acid oxidation in FATP1- and FAT/CD36-overexpressing cells were blunted, confirming that CPT1 activity is necessary for fatty acid import into mitochondria (Fig. 3A, B). We also determined the ratio of incomplete (ASPs) versus complete (CO2) oxidation as a measure of mitochondrial fatty acid oxidation efficiency. The overexpression of CPT1A, FATP1, and FAT/CD36 produced a 30–40% decrease in the ratio ASP/CO2. Importantly, the cooverexpression of CPT1A together with FATP1 or FAT/CD36 produced a 50–60% decrease in this ratio (Fig. 3C), indicating that the overexpression of these proteins increased fatty acid oxidation efficiency.


Novel role of FATP1 in mitochondrial fatty acid oxidation in skeletal muscle cells.

Sebastián D, Guitart M, García-Martínez C, Mauvezin C, Orellana-Gavaldà JM, Serra D, Gómez-Foix AM, Hegardt FG, Asins G - J. Lipid Res. (2009)

Palmitate oxidation in mitochondrial fractions. Mitochondrial fractions that were obtained from cells infected with Ad-LacZ, Ad-FATP1, Ad-CPT1A, or Ad-FAT/CD36 were incubated for 1 h with agitation in 400 μl of pregassed complete MKRH buffer and 50 μl of a 2.5 mM 5:1 palmitate-BSA complex containing 10 μCi/ml [1-14C]palmitic acid, and palmitate oxidation to CO2 (A) and to ASPs (B) was measured. The ratio of ASPs/CO2 was also calculated (C). Data are the mean SE of five experiments performed in duplicate. *P < 0.01 versus Ad-LacZ, **P < 0.001 versus Ad-LacZ, &P < 0.001 versus the overexpression of only one protein (CPT1A, FATP1, or FAT/CD36), and #P < 0.01 versus control without etomoxir.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2724792&req=5

fig3: Palmitate oxidation in mitochondrial fractions. Mitochondrial fractions that were obtained from cells infected with Ad-LacZ, Ad-FATP1, Ad-CPT1A, or Ad-FAT/CD36 were incubated for 1 h with agitation in 400 μl of pregassed complete MKRH buffer and 50 μl of a 2.5 mM 5:1 palmitate-BSA complex containing 10 μCi/ml [1-14C]palmitic acid, and palmitate oxidation to CO2 (A) and to ASPs (B) was measured. The ratio of ASPs/CO2 was also calculated (C). Data are the mean SE of five experiments performed in duplicate. *P < 0.01 versus Ad-LacZ, **P < 0.001 versus Ad-LacZ, &P < 0.001 versus the overexpression of only one protein (CPT1A, FATP1, or FAT/CD36), and #P < 0.01 versus control without etomoxir.
Mentions: To assess the role of FATP1 in fatty acid oxidation, we measured palmitate oxidation in mitochondria-enriched fractions. Palmitate oxidation to CO2 increased 1.7-fold in mitochondria isolated from FATP1-overexpressing cells compared with Ad-LacZ infected cells (0.16 ± 0.009 nmol palmitate/mg protein × h; Fig. 3A). As a positive control, we also evaluated palmitate oxidation in CPT1-overexpressing cells. Overexpression of CPT1A (3-fold increase in protein levels; Fig. 1C), the endogenous CPT1 isoform expressed in these cells (22), produced a 2.6-fold increase in mitochondrial palmitate oxidation to CO2. Moreover, overexpression of FAT/CD36 also increased palmitate oxidation to CO2 by 2.0-fold. To further evaluate the role of FATP1 in fatty acid oxidation, we overexpressed both FATP1 and CPT1 proteins together. The overexpression of CPT1A and FATP1 enhanced fatty acid oxidation to CO2 by 4.7-fold, showing an additive effect (Fig. 3A). Similarly, cooverexpression of CPT1A and FAT/CD36 also produced an additive effect on palmitate oxidation to CO2 (4.1-fold increase). Palmitate oxidation to ASPs, essentially consisting of acyl-carnitines, Krebs cycle intermediates, and acetyl-CoA, was increased from 1.4- to 1.8-fold in CPT1A-, FAT/CD36-, FATP1-, CPT1A+FAT/CD36-, and CPT1A+FATP1-overexpessing cells compared with Ad-LacZ control cells (36.1 ± 1.5 nmol palmitate/mg protein × h; Fig. 3B). When mitochondria were incubated with etomoxir, an irreversible inhibitor of CPT1, the increments in fatty acid oxidation in FATP1- and FAT/CD36-overexpressing cells were blunted, confirming that CPT1 activity is necessary for fatty acid import into mitochondria (Fig. 3A, B). We also determined the ratio of incomplete (ASPs) versus complete (CO2) oxidation as a measure of mitochondrial fatty acid oxidation efficiency. The overexpression of CPT1A, FATP1, and FAT/CD36 produced a 30–40% decrease in the ratio ASP/CO2. Importantly, the cooverexpression of CPT1A together with FATP1 or FAT/CD36 produced a 50–60% decrease in this ratio (Fig. 3C), indicating that the overexpression of these proteins increased fatty acid oxidation efficiency.

Bottom Line: The cooverexpression of FATP1 and CPT1 also enhanced mitochondrial fatty acid oxidation, similar to the cooverexpression of FAT/CD36 and CPT1.However, etomoxir, an irreversible inhibitor of CPT1, blocked all these effects.These data reveal that FATP1, like FAT/CD36, is associated with mitochondria and has a role in mitochondrial oxidation of fatty acids.

View Article: PubMed Central - PubMed

Affiliation: Departments of Biochemistry and Molecular Biology, School of Pharmacy, University of Barcelona, Barcelona, Spain.

ABSTRACT
Carnitine palmitoyltransferase 1 (CPT1) catalyzes the first step in long-chain fatty acid import into mitochondria, and it is believed to be rate limiting for beta-oxidation of fatty acids. However, in muscle, other proteins may collaborate with CPT1. Fatty acid translocase/CD36 (FAT/CD36) may interact with CPT1 and contribute to fatty acid import into mitochondria in muscle. Here, we demonstrate that another membrane-bound fatty acid binding protein, fatty acid transport protein 1 (FATP1), collaborates with CPT1 for fatty acid import into mitochondria. Overexpression of FATP1 using adenovirus in L6E9 myotubes increased both fatty acid oxidation and palmitate esterification into triacylglycerides. Moreover, immunocytochemistry assays in transfected L6E9 myotubes showed that FATP1 was present in mitochondria and coimmunoprecipitated with CPT1 in L6E9 myotubes and rat skeletal muscle in vivo. The cooverexpression of FATP1 and CPT1 also enhanced mitochondrial fatty acid oxidation, similar to the cooverexpression of FAT/CD36 and CPT1. However, etomoxir, an irreversible inhibitor of CPT1, blocked all these effects. These data reveal that FATP1, like FAT/CD36, is associated with mitochondria and has a role in mitochondrial oxidation of fatty acids.

Show MeSH
Related in: MedlinePlus